MOLECULAR BASIS AND USE OF A RADIAL GLIA CELL LINE C6-R

放射状胶质细胞在神经发育过程中短暂出现，在细胞迁移和分化中起关键作用。然而，它们一直很难研究，因为它们在培养中不稳定，而且还没有具有放射状胶质细胞特性的细胞系。我们用突变的磷酸酶受体转染鼠C6胶质瘤细胞系，获得了稳定的细胞系(C6-R)。C6-R具有放射状的形态，在培养和体内均能刺激神经元迁移。虽然C6-R和C6都能在培养中快速增殖，但C6-R不能像C6一样在大鼠脑内形成肿瘤。由于转基因突变受体在许多其他C6克隆中的表达不能解释C6-R和C6之间的表型差异，这种改变很可能是由于在C6-R基因组DNA的Southern杂交中检测到的单个整合位点的遗传中断所致。我们假设，这种整合事件扰乱了促进神经胶质瘤形成或抑制神经胶质生成的基因。这项建议的目标是了解C6-R的细胞特性和这种独特表型的分子基础。此外，鉴于C6-R细胞能够在成熟的CNS中沿白质迁移和排列，并支持神经元迁移，它们将被移植到CNS中，以探索它们在包括损伤后在内的各种情况下促进神经生长的能力。为了了解C6-R在体内形成肿瘤的能力降低的原因，我们将把它的细胞特性，包括增殖和死亡，与亲代C6细胞系进行比较。分子分析将被用于确定导致C6转化为C6-R细胞的基因改变以及导致其表型差异的表达差异。将采用质粒挽救和基因组克隆的方法，从质粒插入的位置恢复DNA。利用C6-R和C6的mRNA进行消减抑制杂交(SSH)，将分析该基因以及其他基因，以确定它们在胶质形成和胶质瘤形成中的作用。这些研究将有助于更好地了解与胶质细胞发育和高级别肿瘤形成有关的基因。对C6-R逆转为体内可形成肿瘤的细胞的研究可能对理解胶质瘤如何发展为高级别恶性肿瘤具有重要意义。最后，放射状神经胶质细胞系的特性可能会产生促进损伤后神经生长的新方法。

Radial glial cells appear transiently during neural development and play key roles in cell migration and differentiation. However, they have been difficult to study because they are unstable in culture and cell lines with radial glial properties have not been available. We have derived a stable cell line (C6-R) from the rat C6 glioma cell line by transfection with a mutant phosphatase receptor. C6-R has radial morphology and stimulates neuronal migration in culture and in vivo. Although both C6-R and C6 can proliferate rapidly in culture, C6-R is unable to form tumors in rat brain like C6. Since expression of the transfected mutant receptor in many other C6 clones can not account for the phenotypic differences between C6-R and C6, it is likely that the alteration is due to genetic disruption at a single integration site that has been detected in Southern blots of C6-R genomic DNA. We hypothesize that this integration event disrupted genes that either promote glial tumor formation or suppress gliogenesis. The goals of this proposal are to understand the cellular properties of C6-R and the molecular basis of this unique phenotype. In addition, given the ability of C6-R cells to migrate and align along white matter in mature CNS and to support neuronal migration, they will be implanted in the CNS to explore their ability to promote growth of nerves in various situations including following injury. To understand the reduced ability of C6-R to form tumors in vivo, its cellular properties, including proliferation and death, will be compared with the parental C6 cell line. Molecular analysis will be directed to identify the genetic alterations responsible for transformation C6 into C6-R cells and the resulting differences in expression that are responsible for their phenotypic differences. Plasmid rescue and genomic cloning methods will be used to recover DNA from the locus of plasmid insertion. Genes at this locus, and other identified by subtractive suppression hybridization (SSH) using mRNA from C6-R and C6 will be analyzed to determine their roles in gliogenesis and formation of gliomas. These studies will lead to a better understanding of genes that are involved in glial development and in formation of high grade tumors. Studies of the reversion of C6-R to cells that can form tumors in vivo may have implications for understanding how gliomas progress to high grade malignancies. Finally, characterization of radial glial cell lines may yield new methods for promoting nerve growth following injury.